The general term cholinesterase (ChE) refers to a family of enzymes involved in nerve impulse transmission. Cholinesterase-inhibiting substances such as organophosphate compounds or carbamate insecticides or drugs prevent the breakdown of acetylcholine, resulting in a buildup of acetylcholine, thereby causing hyperactivity of the nervous system. When humans breathe or are otherwise exposed to these compounds, which has led to the development of these compounds as “nerve gases” or chemical warfare agents.
Those enzymes which preferentially hydrolyze other types of esters such as butyrylcholine, and whose enzymatic activity is sensitive to the chemical inhibitor tetraisopropylpyrophosphoramide (also known as iso-OMPA), are called butyrylcholinesterases (BChE, EC 3.1.1.8).
Butyrylcholinesterase (BChE), also known as plasma, serum, benzoyl, false, or Type II ChE, has more than eleven isoenzyme variants and preferentially uses butyrylcholine and benzoylcholine as in vitro substrates. BChE is found in mammalian blood plasma, liver, pancreas, intestinal mucosa, the white matter of the central nervous system, smooth muscle, and heart. BChE is sometimes referred to as serum cholinesterase as opposed to red cell cholinesterase (AChE).
The use of cholinesterases as pre-treatment drugs has been successfully demonstrated in animals, including non-human primates. For example, pretreatment of rhesus monkeys with fetal bovine serum-derived AChE or horse serum-derived BChE protected them against a challenge of two to five times the LD50 of pinacolyl methylphosphonofluoridate (soman), a highly toxic organophophate compound used as a war-gas [Broomfield, et al. J. Pharmacol. Exp. Ther. (1991) 259:633-638; Wolfe, et al. Toxicol Appl Pharmacol (1992) 117(2):189-193]. In addition to preventing lethality, the pretreatment prevented behavioral incapacitation after the soman challenge, as measured by the serial probe recognition task or the equilibrium platform performance task. Administration of sufficient exogenous human BChE can protect mice, rats, and monkeys from multiple lethal-dose organophosphate intoxication [see for example Raveh, et al. Biochemical Pharmacology (1993) 42:2465-2474; Raveh, et al. Toxicol. Appl. Pharmacol. (1997) 145:43-53; Alton, et al. Toxicol. Sci. (1998) 43:121-128]. Purified human BChE has been used to treat organophosphate poisoning in humans, with no significant adverse immunological or psychological effects (Cascio, et al. Minerva Anestesiol (1998) 54:337).
In addition to its efficacy in hydrolyzing organophosphate toxins, there is strong evidence that BChE is the major detoxifying enzyme of cocaine [Xie, et al. Molec. Pharmacol. (1999) 55:83-91]. Cocaine is metabolized by three major routes: hydrolysis by BChE to form ecgonine methyl ester, N-demethylation from norcocaine, and non-enzymatic hydrolysis to form benzoylcholine. Studies have shown a direct correlation between low BChE levels and episodes of life-threatening cocaine toxicity. A recent study has confirmed that a decrease of cocaine half-life in vitro correlated with the addition of purified human BChE.
In view of the significant pharmaceutical potential of ChE enzymes, research has focused on development of recombinant methods to produce them. Recombinant enzymes, as opposed to those derived from plasma, have a much lower risk of transmission of infectious agents, including viruses such as hepatitis C and HIV.
The cDNA sequences have been cloned for both human AChE (see U.S. Pat. No. 5,595,903) and human BChE [see U.S. Pat. No. 5,215,909 to Soreq; Prody, et al. Proc. Natl. Acad. Sci. USA (1987) 84:3555-3559; McTiernan, et al. Proc. Natl. Acad. Sci USA (1987) 84:6682-6686]. The amino acid sequence of wild-type human BChE, as well as of several BChE variants with single amino acid changes, is set forth in U.S. Pat. No. 6,001,625.
Notably, none of the recombinant expression systems reported to date have the ability to produce BChE in quantities sufficient to allow development of the enzyme as a drug to treat such conditions as organophosphate poisoning, post-surgical apnea, or cocaine intoxication. However, an additional problem is longevity. Thus, the longer the BChE remains in the system of a person treated, the longer it is available for detoxification. Such lifespan is referred to as the “mean residence time” (MRT) in the system.
The current state of art for BChE is directed to making the tetramer form because it is the “native form” and is thus considered to be more stable with a longer “mean residence time” (MRT). However, due to the very large size of the tetramer, it is difficult to prepare. In addition, such preparation usually results in a mixture of tetramer, dimer and monomer forms with low yield. Such preparation has proven both very cumbersome and very expensive to purify and characterize. As a result, it is probably too expensive to make as a useful therapeutic product. In view of the foregoing, more powerful methods of producing BChE are needed.
In sum, the current obstacles in the manufacture of the native BChE molecule as a bioscavenger product which are: 1) low yield, 2) complex manufacturing process (milk), 3) short half-life (thus requiring pegylation), 4) highly heterogeneous product (difficult to characterize and obtain FDA approval) and 5) high cost of the product.
The present invention addresses at least some of these problems by providing inter alia a truncated monomeric form of BChE. While the the monomer form is just as active as the tetrameric form, it has been considered to be less stable (i.e., have a lower “MRT”) than the tetramer. This may be because the protein made is not properly glycosylated and/or sialylated. Applicants have identified a cell line and clone to accomplish this result. Furthermore, if the full length BChE is made, the cells produce a mixture of monomer, dimer and tetramer so that the present invention also provides a means of producing preferably the monomeric form.